This Project will continue to provide the entire PPG with an increasingly integrative structural context for the proposed studies that seek an understanding of the functional mechanisms of the neurotransmitter transporters (NTs), and of interactions with cellular components that regulate their function and integrate it into the cell signaling processes. The mechanisms of interest underiie observable pharmacological properties and physiological effects of drugs of abuse, and make these NTs both a target for the design of therapeutic drugs, and a key element in (i)-the determinants, (ii)-the effects, and (iii)-the undesirable consequences, of substance abuse. We will use computational modeling and simulation to determine the conformational changes in the TM domains of NTs that are involved in the allosteric coupling between ligand and ion transport in DAT and other NT, and the changes produced by the psychostimulants amphetamine and cocaine, based on the hypothesis that the allosteric mechanism consist of a conserved spatial network of interactions among residues positioned non-sequentially in the transporters, which are triggered in a defined temporal sequence identifiable from the SMD simulations and extensive MD equilibrations of the resulting intermediate states. We also aim for a mechanistic characterization of the N- and C-terminal segments ofthe NTs DAT and SERT, in different functional states ofthe proteins (e.g, as induced by drugs of abuse), based on a hypothesis that functional roles of these segments depend on (i)-specific conformations they adopt in different functional states, and (ii)-modulation of these conformations by cellular processes involving phosphorylation, and/or (scaffold and adaptor) protein binding. We will model and simulate computationally the functional determinants of such scaffold, adaptor and membrane remodeling domains that interact with the NTs in mechanisms of signaling and trafficking in the membrane environment. To this end will study biophysical properties, mechanisms of oligomerization, and dynamic regulation of several protein families including (i) PDZ-containing multidomain proteins (e.g., PICK1); (ii) BAR domains and their complexes with specific membrane regions; (iii) membrane-inserted proteins such as Flotillin 1.